Transcriptome profiling of purified mouse platelets from Nxf1 mutant and control mice [Platelet]

Purpose: Nxf1 is thought to be an essential nuclear exporter of messenger RNA (mRNA) in eukaryotic cells. Whether perturbations in the Nxf1 pathway affect mammalian physiology is not known. The aim of this study is to determine the impact of a Nxf1 mutation in the representation of mRNA transcripts in platelets. Methods: Platelets were purified from individual males. Blood was obtained by cardiac puncture into 0.1 volume of Aster Jandl citrate-based anticoagulant. Mouse platelet rich fraction was obtained by centrifugation of the murine blood at 125 g for 8 min at room temperature, followed by centrifugation of the supernatant buffy coat at 125 g for 8 min. Mouse platelets were washed by two sequential centrifugations at 860 g for 5 min in 140 mM NaCl, 5 mM KCl, 12 mM trisodium citrate, 10 mM glucose, and 12.5 mM sucrose, pH 6.0.The platelet pellet was resuspended in 10 mM Hepes, 140 Mm NaCl, 3 mM KCl, 0.5 mM MgCl2, 10 mM glucose, and 0.5 mM NaHCO3, pH 7.4. The purity of each platelet suspension was assessed by flow cytometry and suspensions for which more than 98% of total events were CD41+ platelets were pooled together. RNA was purified with Norgen cytoplasmic and nuclear fractionation RNA purification kit. Platelets were purified from individual Nxf1 mutant males (littermate controls are available from GEO accession GSE75896). The purity of each suspension was assessed by flow cytometry. Suspensions of identical genotype for which more than 98% of total events were CD41+ platelets were pooled together. Pooled platelet preparations from mutant (this study) and wild-type littermate controls (GSE75896) were prepared on 3 distinct days. The GSM4196370-GSM4196372 sample records are duplicated sample records of GSM1969530-GSM1969532 (in GSE75896) for the convenient retrieval of the complete raw data from SRA.研究目的：Nxf1被认为是真核细胞中信使RNA（mRNA）的核心核输出蛋白。目前尚不清楚Nxf1通路的扰动是否会对哺乳动物生理机能产生影响。本研究旨在明确Nxf1突变对血小板内mRNA转录本表达特征的影响。 实验方法：从雄性个体中分离纯化血小板。通过心脏穿刺采集血液，将血液加入0.1倍体积的Aster Jandl枸橼酸盐抗凝剂中。小鼠富血小板组分的制备步骤如下：室温下以125 g离心小鼠全血8分钟，取上清液的棕黄层，以125 g再次离心8分钟。使用洗涤缓冲液（含140 mM氯化钠、5 mM氯化钾、12 mM枸橼酸钠、10 mM葡萄糖及12.5 mM蔗糖，pH 6.0）对小鼠血小板进行两次连续的860 g、5分钟离心洗涤。将洗涤后的血小板重悬于重悬缓冲液（含10 mM羟乙基哌嗪乙磺酸（Hepes）、140 mM氯化钠、3 mM氯化钾、0.5 mM氯化镁、10 mM葡萄糖及0.5 mM碳酸氢钠，pH 7.4）中。 采用流式细胞术检测每份血小板悬液的纯度，将总检测事件中98%以上为CD41阳性血小板的悬液合并。使用Norgen细胞质与细胞核组分分离RNA纯化试剂盒提取RNA。 本研究的血小板样本取自Nxf1突变雄性个体，其野生型同窝对照数据可从GEO登录号GSE75896获取。同样通过流式细胞术检测突变型血小板悬液的纯度，合并总检测事件中98%以上为CD41阳性血小板的同基因型悬液。 本研究的突变型血小板样本与野生型同窝对照样本（对应GSE75896数据集）均于3个独立实验日完成制备。其中GSM4196370至GSM4196372的样本记录为GSE75896中GSM1969530至GSM1969532的重复样本，以便于从序列读取档案（Sequence Read Archive, SRA）中便捷获取完整原始数据。